Reverse transcription is the process by which a retrovirus such as HIV-1 converts its genetic material (single-stranded RNA) into a double-stranded DNA copy that is integrated into host chromosomal DNA. This process is complex and is catalyzed by the virion-associated enzyme, reverse transcriptase (RT). However, another viral protein, the nucleocapsid protein (NC), is also required for efficient and specific viral DNA synthesis. (A) We study the mechanistic basis for NC activity. HIV-1 NC is a small, basic nucleic acid binding protein with two zinc fingers, each containing the invariant CCHC zinc-coordinating motifs. It is a nucleic acid chaperone, i.e., NC has the ability to catalyze conformational rearrangements that result in formation of the most thermodynamically stable nucleic acid structures. This property is critical for promoting the two strand transfer events that are needed for synthesis of full-length plus- and minus-strand viral DNA. In minus-strand transfer, the initial product of reverse transcription, (-) strong stop DNA, is translocated to the 3-prime end of viral RNA (termed acceptor RNA) in a reaction facilitated by base-pairing of the complementary repeat regions (containing the highly structured TAR stem-loops), which are present at the ends of the RNA and DNA partners. (i) Recent studies have focused on the effect on the nucleic acid chaperone activity of NC when it is embedded in Gag (the precursor to the viral structural proteins) and partially processed Gag proteins. Activity was assayed using a reconstituted minus-strand transfer assay system, a highly sensitive read-out for chaperone function. Our experiments demonstrate that chaperone activity resides in the NC domain of the precursor proteins. Moreover, we now report for the first time that Gag and Gag-derived proteins have annealing and helix destabilizing activity, since these proteins stimulate minus-strand transfer. Surprisingly, unlike NC, high concentrations of Gag proteins block the DNA elongation step in strand transfer. This result is consistent with nucleic acid-driven multimerization of Gag and the reported slow dissociation of Gag from bound nucleic acid, which prevent RT from traversing the template (roadblock mechanism). These findings illustrate one reason why NC (and not Gag) has evolved as critical cofactor in reverse transcription. Additionally, the ability of Gag to act as roadblock to DNA polymerization could help to prevent premature viral DNA synthesis prior to protease cleavage of the precursor. (ii) We have also shown that NC duplex destabilization activity together with RNase H cleavage block mispriming by non-polypurine tract RNAs during initiation of plus-strand DNA synthesis. These findings demonstrate a previously unrecognized role for NC in selection of the correct primer and ensuring the fidelity of plus-strand initiation. (B) Our interest in host proteins that might affect HIV-1 reverse transcription has led us to investigate human APOBEC3G (A3G), a cellular cytidine deaminase with two zinc finger domains, which blocks HIV-1 reverse transcription and replication in the absence of the viral protein known as Vif (virus infectivity factor). The antiviral effect has been shown to be largely deaminase-dependent, but there is also a deaminase-independent component. (i) One goal of our A3G studies has been to elucidate the mechanism for A3G inhibition of reverse transcription. Using purified proteins, we have investigated the interplay between A3G, NC, and RT in reconstituted reactions representing individual steps in the reverse transcription pathway. We have reported that A3G does not affect the kinetics of NC-mediated annealing or the RNase H activity of RT. In sharp contrast, A3G significantly inhibits all RT-catalyzed elongation reactions with or without NC and without a requirement for A3G catalytic activity. Data from single-molecule DNA stretching analyses support an unusual mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC, and RT. Thus, like Gag, A3Gs slow dissociation from bound nucleic acid also results in a roadblock to DNA polymerization. (ii) The Vif protein abrogates A3G function by mediating degradation of A3G through the ubiquitination/proteosome pathway. In a recent collaborative study, we focused on the lysine residues that are critical for A3G binding to Vif and subsequent recruitment of A3G to the ubiquitin ligase complex. Using structure-guided mutagenesis, we identified four critical lysine residues in the C-terminal domain of A3G. Interestingly, in our model, these residues cluster at sites that are opposite to the N-terminal Vif-interaction region of the protein. This information will be valuable for designing antiviral therapeutic strategies. (iii) In current work, we studying the human APOBEC3A (A3A) protein, which has only one zinc finger domain and is a potent inhibitor of retrotransposition by Line-1 and Alu non-LTR elements. We found that A3A can be expressed in E. coli and purified from bacterial extracts. Recently, conditions were found that yield large amounts of purified protein for detailed biochemical and structural analysis, which is now in progress. The biological activity of wild-type A3A and mutant constructs is assessed by activity in a Line-1 retrotransposition assay. Structure-guided mutagenesis is used to target residues that should be of greatest interest. (C) Our laboratory has also been investigating the role of the HIV-1 capsid protein (CA) in early postentry events, a stage in the infectious process that is still not completely understood. Our previous studies illuminated the intimate connection between infectivity, proper core morphology, structural integrity of the CA protein, and the ability to undergo reverse transcription. In current work, we are investigating the effect of point mutations in the five residues comprising the hinge region that connects the N- and C-terminal domains of CA and in the two residues that each flank the N- or C-terminal end of the hinge, respectively. Although all of the mutants produce virus particles, only two (within the hinge region itself) have infectivity in a single-cycle replication assay. The lack of infectivity is correlated with the appearance of aberrant cores as observed by transmission electron microscopy, detergent sensitivity of the cores, as well as a defect in reverse transcription following infection. Two of the hinge mutants have an attenuated phenotype. For example, despite being poorly infectious, a significant number of mutant particles (though not all) contain conical cores. Experiments to investigate the properties of these unusual mutants in more detail are in progress. Collectively, the results obtained thus far indicate that perturbation of the CA protein in the hinge region can lead to core instability and distortion of core architecture, with downstream effects on reverse transcription and infectivity.